Project Description (Abstract) Oxidative stress is a state of reduction and oxidation (redox) imbalance caused by increased reactive oxygen species (ROS) generation, decreased antioxidant capacity, or both. Thus, it is important for cells to maintain a state of redox homeostasis by regulating the equilibrium between ROS production and scavenging capacity. Cancer cells are often exposed to intrinsic and extrinsic stresses that produce high levels of ROS and DNA damage. More importantly, we and others have shown that induction of oxidative stress is one of the underlying mechanisms of action for many anticancer drugs and radiation. Growing evidence has revealed that cancer stem cells (CSCs) play a critical role in therapy resistance and tumor relapse, as demonstrated by the fact that CSCs are selectively enriched in residual tumors following anticancer therapies. However, there is still an important gap in our knowledge of the mechanisms whereby CSCs maintain redox balance and counteract ionizing radiation (IR) and ROS-generating drug-induced oxidative stress. Our preliminary studies have demonstrated that CSCs are less responsive to IR and cisplatin-induced cell killing than non-stem cancer cells (NSCs) of human triple-negative breast cancer (TNBC). Moreover, we have found that ROS levels are significantly lower in CSCs than NSCs. These results suggest that CSCs have the capacity to maintain low levels of ROS and thus evade oxidative stress-induced toxicity. On the basis of these findings, we hypothesize that CSCs adopt a distinct redox signaling system to counteract oxidative stress and maintain lower levels of ROS, which consequently confers therapy-resistance to CSCs by overriding oxidative stress-induced cell death. Therefore, targeting of selective redox signaling pathways should sensitize therapy-resistant CSCs to ROS-generating anticancer drugs and radiotherapy. Three specific Aims are proposed to test this hypothesis: Aim 1 will define differences in redox signaling pathways between CSCs and NSCs; Aim 2 will elucidate the mechanisms whereby CSCs maintain lower levels of ROS and self-renewal capacity; and Aim 3 will determine if targeting of selective redox signaling pathways sensitizes therapy-resistant CSCs to chemotherapy and radiation. We anticipate the outcomes of this project will provide novel insights into the mechanisms whereby CSCs evade anticancer therapy-induced oxidative stress. New information obtained from this project will be essential to the development of innovative therapeutic strategies to eliminate therapy-resistant CSCs in human cancers and thus improve the efficacy of cancer treatment.